It is desirable to provide rapid detection, identification, and/or enumeration of microorganisms in biological and nonbiological samples. While a number of tests have been developed for a variety of markets using a number of widely varying methodologies, there continues to be a need for simple, rapid, inexpensive, sensitive, and specific detection tests. Examples of presently used tests are DNA probes, immunoassays, electrical measurements, ATP measurements, and enzymatic substrate tests. Currently, the most sensitive tests do not readily detect less than 10.sup.5 cfu/ml. Since bacteria are not readily found in those concentrations in food, clinical, environmental, etc. samples, most tests require an overnight pre-enrichment step using conventional culture media to allow the sufficient growth and division of these microorganisms. In other words, a sample is taken and must be cultured in order to allow growth and multiplication of the microorganisms in the sample to a quantity detectable by present day technology.
New emerging detection methods for bacteria have greater predicted sensitivity than those presently on the market. These technologies are predicting sensitivities of 10.sup.1 -10.sup.3 cfu/ml. As a result, these technologies can considerably shorten but not totally eliminate the pre-enrichment step. Since none of these methods can address the diverse market needs, and as of yet have not been proven in the marketplace, new additions to these methods and further improvements of the existing methods are still in need.
An example of an enzymatic substrate test is the "MUG Test" that was first proposed in 1982 by Feng et al, "Fluorogenic assays for immediate confirmation of Escherichia coli", Applied and Environmental Microbiology, Vol. 43, 1982, pp. 1320-1329. The MUG test facilitated the detection of E. coli, although it improved total coliform detection very little.
Edberg and collaborators modified the "MUG Test" by devising a minimal culture medium, similar to those used for many years by bacterial geneticists. Edberg, S. C. et al, "National Field Evaluation of a Defined Substrate Method for the Simultaneous Enumeration of Total Coliforms and Escherichia coli from Drinking Water," Applied and Environmental Microbiology, Vol. 54, 1988, pp. 1595-1601, correction p. 3197. In this technology, the culture medium contains little in the way of organic nutrients except for 4-methylumbelliferyl-b-D-glucuronide (MUG) and a second substrate, o-nitrophenyl-b-D-galactoside (ONPG).
The problem with E. coli detection based on MUG hydrolysis is that the glucuronidase gene (uid A) seems to be present in the E. coli, but is not expressed in many of the strains, including some of the pathogenic strains. Feng, et al, "Presence of b-D-Glucuronidase Gene Sequences in MUG Assay (-) Escherichia coli", Abstracts of the 90th Annual Meeting of the American Society for Microbiology, 1990, Abstract Q-7, p. 289; Hartman, P. A., "The MUG (Glucuronidase) Test for Escherichia coli in Food and Water," in A. Balows, R. C. Tilton, and A. Turano, Ed., Rapid Methods and Automation in Microbiology and Immunology, Brixia Academic Press, Brescia, Italy, 1989, pp. 290-308. In some media, inhibition of glucuronidase synthesis by lactose may occur, but other factors which are currently not totally understood may also be involved.
Others have reported various other methods of detecting and identifying E. coli from bacterial colonies. Edberg et al in "Comparison of--glucuronidase-based substrate systems for identification of Escherichia coli, J. Clin. Microbiology, Sept. 1986, pp. 368-371, discloses a method based on the measurement of beta glucuronidase which is claimed to be specific and inexpensive for the identification of E. coli. Restaino et al in "Use of Chromogenic Substrate 5-bromo-4-chloro-3-indolyl-B-D-glucuronide (X-GLUC) for enumerating Escherichia coli in 24 H from ground beef, J. Food Protection, Vol., 53, June 1990, pp. 508-510 discloses a 24 hour direct plating method for E. coli using the X-GLUC incorporated into a peptone-tergitolagar base.
These present methodologies for coliform and E. coli detection are marginal in performance.
The present invention recognizes that growing bacterial cells make use of approximately 1000 to 5000 enzymatically catalyzed reactions. Hydrolases constitute one of the largest groups of enzymes present in microorganisms. A strong correlation exists between the growth of bacteria and the detection of hydrolytic enzyme activity. As a result of this correlation, these enzymes have been extensively utilized to determine the presence and/or to identify microorganisms in biological and nonbiological samples. This approach to the detection of microorganisms is limited by the number of cells and their physiological state, sensitivity of the assay methods, types of substrates, and the enzymes being measured. Assay methods used are, for example, colorimetric, turbidometric, radiometric, fluorometric, and photometric.
Estimates of the cellular content of several proteins indicate that on the average 10.sup.-20 to 10.sup.-19 mole of any specific protein or enzyme is present per actively growing bacterial cell. Photometric measurements obtained with 1, 2-dioxetane chemiluminescent substrates of the enzymes alkaline phosphatase and beta-D-galactosidase detect 10.sup.-21 and 10.sup.-19 mole of these enzymes, respectively. Thus, it is theoretically possible to detect 1-100 cfu/ml of a sample with chemiluminescent 1,2-dioxetane enzyme substrates.
The U.S. Pat. No. 4,857,652 to Schaap, issued Aug. 15, 1989, discloses novel light producing 1,2-dioxetanes. These chemiluminescent compounds can be triggered by an activating agent to generate light. This mechanism for light production involves two steps. Step one involves the thermal or the enzyme catalyzed decomposition of a high energy material (generally a peroxide) yielding one of the reaction products in a triplet or singlet electronic excited state. The second step is the emission of a photon (fluorescence or phosphorescence) from this excited species producing the light observed from the reaction. As set forth by Schaap et al in Clinical Chemistry, Vol. 35, No. 9, 1989 (1863-1864), more than 350 papers have described investigations of these high energy peroxides which undergo spontaneous decomposition to generate chemiluminescence. Schaap et al developed new dioxetanes that are thermally very stable but can be chemically and enzymatically triggered to produce chemiluminescence on demand. Chemical or enzymatic removal of the protecting group from the stable dioxetane produces an unstable aryloxide dioxetane which decomposes to provide the observed chemiluminescence. This can be made pH dependent such that an enzyme can cause the stable form to become unstable in a neutral pH but light emission through energy transfer is only completed through changing the pH to an alkaline state. This change in pH is referred to as an enhancer system which provides a 400 fold increase in the chemiluminescence efficiency of the reaction in the presence of the enzyme alkaline phosphatase. Schaap et al recognized that the luminescent reaction can be used for ultrasensitive detection of phosphatase-linked antibodies and DNA probes. For example, the Photo Gene.TM. system manufactured by Life Technologies, Inc. of Gaithersburg, Md., utilizes such a chemiluminescent reaction for the detection of nucleic acids. Up until this time, these uses of the chemiluminescent dioxetanes have been limited to the detection of DNA probes and alkaline phosphatase-linked antibodies.
The present invention utilizes chemiluminescent compounds, such as 1,2-dioxetane derivatives, for the detection and identification of microorganisms in biological and nonbiological samples.